Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus

The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, has been determined from 2D H-1 NMR data, Robustoxin is a polypeptide of 42 residues cross-linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1-15, 8-20, 14-31 and 16-42 (a 1-4/2-6/3-7/5-8 pattern), The structure consists of a small three-stranded, anti-parallel beta-sheet and a series of interlocking gamma-turns at the C-terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the omega-conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non-polar residues, Both of these structural features may play a role in its binding to the voltage-gated sodium channel. (C) 1997 Federation of European Biochemical Societies.

Hypoechinorhynchus robustus sp n. from Notolabrus parilus (Labridae) from Western Australia with a discussion on the validity of the hypoechinorhynchidae (Acanthocephala : Palaeacanthocephala)

Hypoechinorhynchus robustus sp. n. is described from Notolabrus parilus (Richardson) (Labridae) from Pt Peron, Western Australia. It has a proboscis with 30 hooks arranged in ten longitudinal rows: 5 rows of a small apical spine, a large anterior hook and a small posterior spine, 5 rows of a large anterior hook, a middle spine and a posterior spine. The new species is distinguished from other species of the genus by having a set of 5 small apical spines anterior to the large hooks on the proboscis, by having lemnisci that barely extend beyond the proboscis receptacle and testes which are more adjacent than tandem. H. robustus also has robust trunk spines anteriorly. Re-examination of Hypoechinorhynchus alaeopis Yamaguti, 1939 (type species) revealed trunk spines that had been overlooked previously. The Hypoechinorhynchidae is made a junior synonym of Arhythmacanthidae because there is considerable overlap between the two family diagnoses, particularly in that both families have a proboscis armature that changes abruptly from small basal spines to large apical (or subapical if present) hooks. The genus Hypoechinorhynchus is placed in the subfamily Arhythmacanthinae because it has trunk spines and a spherical proboscis with few hooks (relative to other arhythmacanthid genera). It is also proposed that Heterosentis magellanicus (Szidat, 1950) be returned to the genus Hypoechinorhynchus since it was transferred to Heterosentis primarily because it had trunk spines. The other hypoechinorhynchid genus contained only Bolborhynchoides exiguus (Achmerov et Dombrowskaja-Achmerova, 1941) Achmerov, 1959 and is relegated to incertae sedis.

Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

Influência da temperatura e umidade no desenvolvimento ninfal de Rhodnius robustus

OBJETIVO: Avaliar as diferenças significativas de desenvolvimento ninfal da espécie Rhodnius robustus Larrousse, 1927, submetida a diferentes condições de temperatura e umidade, que se encontra distribuída na região Norte do Brasil (Acre, Amazonas e Pará) e na Colômbia, no Equador, no Peru e na Venezuela. MÉTODOS: Formaram-se três grupos de Rhodnius robustus, mantidos em diferentes condições de laboratório: 33/40 (33+/-1°C e 40+/-5% UR -- umidade relativa), 33/70 (33+/-1°C e 70+/-5% UR) e 28/70 (28+/-1°C e 70+/-5% UR), para observar o período de incubação dos ovos, o desenvolvimento de cada estádio, os percentuais de mortalidade e os número de repastos realizados. RESULTADOS: O menor período médio de desenvolvimento ninfal foi observado no grupo 28/70 com as médias: 14,4; 17,3; 20,3; 22,8 e 40. O desenvolvimento embrionário apresentou diferenças significativas entre os tratamentos utilizados (p<0,01). O número de repastos teve aumento gradual com a aproximação da fase adulta em todos os grupos testados, exceto no terceiro estádio do grupo 33/70. Os menores percentuais de mortalidade foram os do grupo 28/70. CONCLUSÕES: Temperaturas elevadas, independentes das umidades associadas, podem acelerar o ciclo biológico da espécie. Entretanto, elas podem prejudicar a sobrevivência das colônias, impedindo sua manutenção em laboratório.

Perfil de la mortalidad de hembras de Rhodnius robustus (Heteroptera: Reduviidae)

Se estudió la mortalidad de 48 hembras adultas de Rhodnius robustus. Dada la significancia estadística del ajuste de los datos al modelo de Gompertz (p<0.0001), se puede afirmar que el mismo permite describir la mortalidad de esta especie.

Susceptibilidade de Rhodnius neglectus, Rhodnius robustus e Triatoma infestans (Hemiptera, Reduviidae, Triatominae) à infecção por duas cepas de Trypanosoma cruzi (Kinetoplastidae, Trypanosomatidae) utilizando xenodiagnóstico artificial

A susceptibilidade de ninfas de 3º estádio de Rhodnius neglectus, R. robustus e Triatoma infestans às cepas Y e AMJM de Trypanosoma cruzi foi verificada utilizando xenodiagnóstico artificial. Para a leitura do xenodiagnóstico, as fezes dos triatomíneos foram examinadas a cada dois dias, a partir do 5º até o 31º dia pós infecção, pela técnica de compressão abdominal. Os resultados mostraram diferenças na susceptibilidade dos triatomíneos para as duas cepas estudadas e o período ótimo de leitura variou do 11º ao 19º dias para a cepa Y e do 11º ao 15º dias para a cepa AMJM. Também, pôde-se concluir que para a cepa Y, as três espécies de triatomíneos demonstraram boa susceptibilidade, enquanto para a cepa AMJM, a melhor susceptibilidade foi observada com R. neglectus, seguida pelo T. infestans e R. robustus.

Biologia comparativa e comportamento alimentar de Rhodnius neglectus e Rhodnius robustus (Triatominae) sob condições de laboratório

INTRODUÇÃO: A competência vetorial de triatomíneos é determinada a partir de estudos sobre biologia e comportamento alimentar em condições de campo e/ou laboratório. Fatores como número de picadas, quantidade de sangue ingerido e tempo de defecação têm implicações na transmissão de tripanosomatídeos. Parâmetros biológicos e comportamentais de Rhodnius neglectus e R. robustus foram comparados experimentalmente para estimar diferenças no potencial de transmissão de tripanosomatídeos. MÉTODOS: Os triatomíneos foram observados diariamente para determinar o período de desenvolvimento ninfal, mortalidade, detecção da fonte alimentar, número de picadas, tempo do repasto sanguíneo, quantidade de sangue ingerido, tempo entre o final do repasto e a primeira defecação e frequência de defecação. RESULTADOS: Apesar do período ninfal de R. neglectus (156,4 ± 25,05d) ter sido menor que o de R. robustus (204,7 ± 13,22d), a mortalidade foi similar entre as espécies (63,8 e 65%, respectivamente). R. robustus e R. neglectus detectaram rapidamente a fonte alimentar, especialmente no primeiro estádio (2,5 e 1,6min, respectivamente). Apesar do tempo de repasto sanguíneo ter sido similar entre as espécies, R. robustus ingeriu em média uma maior quantidade de sangue em todos os estádios, com maiores valores para as ninfas V. As ninfas de R. neglectus picaram mais vezes as fontes alimentares, defecaram mais rápido e mais frequentemente que as de R. robustus. CONCLUSÕES: Sob as condições de laboratório usadas, R. neglectus possui um maior potencial para transmissão de Trypanosoma cruzi e T. rangeli que R. robustus, atributo que deve ser avaliado em infecções experimentais.

Dinâmica reprodutiva de Oligosarcus jenynsii e O. robustus (Characiformes, Characidae) na lagoa Fortaleza, Rio Grande do Sul, Brasil

Oligosarcus jenynsii (Günther, 1864) and O. robustus Menezes, 1969 are fish species distributed in Rio Grande do Sul, Brazil, Uruguay, and northern Argentina. The reproductive period and recruitment, sex ratio, absolute and relative fecundity, and body length at first gonadal maturation of the two carnivorous species from Fortaleza Lagoon were analized. The specimens were sampled monthly, from May 2000 to April 2001, with fishing effort of 24 hours/month, using stationary gillnets of different mesh sizes and seine net (three samples per edge). The records of each individual included total length, total weight, gonad weight, sex and gonadal maturity stage. The reproductive period of both O. jenynsii and O. robustus ranges from May/June to November/December, according to the bimonthly variation of the mean values of gonosomatic index, and the relative frequencies of the gonadal maturation stages. Recruitment of new individuals to the population occurs from November/December to March/April. The sex ratio is different from 1:1 for O. jenynsii and similar to 1:1 for O. robustus. The mean absolute fecundity, calculated by counting sub-sampled oocytes from mature females, was 14,483 oocytes for O. jenynsii, and 16,308 oocytes for O. robustus. The first maturation curve shows that O. jenynsii begins to reproduce between 84 mm and 104 mm (total length), and O. robustus between 126 mm and 146 mm, probably at similar ages.

Chagas' disease in the Amazon Basin: V. Periurban palms as habitats of Rhodnius robustus and Rhodnius pictipes - triatomine vectors of Chagas' disease

Trypanosoma cruzi infected Rhodnius robustus and/or Rhodnius pictipes were commonly found, in large numbers, in the Brazilian Amazonian palms Maximiliana regia ("inajá"), Acrocomia sclerocarpa ("mucajá") and Orbignya speciosa ("babaçu"). The common opossum, Didelphis marsupialis, was the animal most frequently associated with triatomine infested palms. R. pictipes, frequently light-attracted into houses from palm trees, was the probable source of an acute case of Chagas' disease in the vicinity of Belém. It is considered that triatomine infested palms are likely to cause some cases of acute Chagas' disease in the States of Amazonas and Rondônia. Possible control methods are suggested.

Rhodnius robustus in Bolivia identified by its wings

Wings of a Rhodnius specimen from Alto Beni (Bolivia) was examined for identification and compared with R. stali, R. robustus, (certified Bolivian species), R. pictipes and R. prolixus (suspected Bolivian species). A projection of the unidentified wings as supplementary data into a discriminant analysis of shape revealed clear cut differences with R. stali and R. pictipes, less differences with R. prolixus, and none with R. robustus. Combining global size and shape of the wings, the unknown specimen was identified as R. robustus. Thus, this study confirmed the presence of R. robustus in Bolivia. It also highlighted the possibility of morphometrics to taxonomically interpret one individual, or even one piece of an individual, when related species data are available for comparison.

Susceptibilidade de Rhodnius neglectus, Rhodnius robustus e Triatoma infestans (Hemiptera, Reduviidae, Triatominae) à infecção por duas cepas de Trypanosoma cruzi (Kinetoplastidae, Trypanosomatidae) utilizando xenodiagnóstico artificial

A susceptibilidade de ninfas de 3º estádio de Rhodnius neglectus, R. robustus e Triatoma infestans às cepas Y e AMJM de Trypanosoma cruzi foi verificada utilizando xenodiagnóstico artificial. Para a leitura do xenodiagnóstico, as fezes dos triatomíneos foram examinadas a cada dois dias, a partir do 5º até o 31º dia pós infecção, pela técnica de compressão abdominal. Os resultados mostraram diferenças na susceptibilidade dos triatomíneos para as duas cepas estudadas e o período ótimo de leitura variou do 11º ao 19º dias para a cepa Y e do 11º ao 15º dias para a cepa AMJM. Também, pôde-se concluir que para a cepa Y, as três espécies de triatomíneos demonstraram boa susceptibilidade, enquanto para a cepa AMJM, a melhor susceptibilidade foi observada com R. neglectus, seguida pelo T. infestans e R. robustus.

Estudo de transcriptomas por RNAseq em tecidos de cabeça e glândula salivar de Rhodnius montenegrensis e Rhodnius robustus (Hemiptera, Reduviidade, Triatominae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Chemical contaminants in juvenile gray whales (Eschrichtius robustus) from a subsistence harvest in Arctic feeding grounds

Gray whales are coastal migratory baleen whales that are benthic feeders. Most of their feeding takes place in the northern Pacific Ocean with opportunistic feeding taking place during their migrations and residence on the breeding grounds. The concentrations of organochlorines and trace elements were determined in tissues and stomach contents of juvenile gray whales that were taken on their Arctic feeding grounds in the western Bering Sea during a Russian subsistence harvest. These concentrations were compared to previously published data for contaminants in gray whales that stranded along the west coast of the US during their northbound migration. Feeding in coastal waters during their migrations may present a risk of exposure to toxic chemicals in some regions. The mean concentration (standard error of the mean, SEM) of Σ PCBs [1400 (130) ng/g, lipid weight] in the blubber of juvenile subsistence whales was significantly lower than the mean level [27 000 (11 000) ng/g, lipid weight] reported previously in juvenile gray whales that stranded in waters off the west coast of the US. Aluminum in stomach contents of the subsistence whales was high compared to other marine mammal species, which is consistent with the ingestion of sediment during feeding. Furthermore, the concentrations of potentially toxic chemicals in tissues were relatively low when compared to the concentrations in tissues of other marine mammals feeding at higher trophic levels. These chemical contaminant data for the subsistence gray whales substantially increase the information available for presumably healthy animals.

Gray Whales (Eschrichtius robustus) Off Sakhalin Island, Russia: Seasonal And Annual Patterns Of Occurrence

The annual return, seasonal occurrence, and site fidelity of Korean-Okhotsk or western gray whales on their feeding grounds off northeastern Sakhalin Island, Russia, were assessed by boat-based photo-identification studies in 1994-1998. A total of 262 pods were observed, ranging in size from 1 to 9 whales with an overall mean of 2.0'. Sixty-nine whales were individually identified, and a majority of all whales (71.0%) were observed in multiple years. Annual sighting frequencies ranged from 1 to 18 d, with a mean of 5.4 d. The percentage of whales re-identified from previous years showed a continuous annual increase, reaching 87.0% by the end of the study. Time between first and last sighting of identified individuals within a given year was 1-85 d, with an overall mean of 40.6 d. Annual calf proportions ranged from 4.3% (1997) to 13.2% (1998), and mother-calf separations generally occurred between July and September. The seasonal site fidelity and annual return of whales to this part of the Okhotsk Sea emphasize its importance as a primary feeding ground for this endangered population.